Subscriber access provided by NEW YORK UNIV
Article
Selective 1D TOCSY NMR Experiments for a Rapid Identification of Minor Components in the Lipid Fraction of Milk and Dairy Products: Towards Spin-Chromatography? Christina Papaemmanouil, Constantinos G. Tsiafoulis, Dimitrios Alivertis, Ouranios Tzamaloukas, Despoina Miltiadou, Andreas Tzakos, and Ioannis P. Gerothanassis J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b01335 • Publication Date (Web): 19 May 2015 Downloaded from http://pubs.acs.org on May 26, 2015
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 26
Journal of Agricultural and Food Chemistry
1
Selective 1D TOCSY NMR Experiments for a Rapid Identification of
2
Minor Components in the Lipid Fraction of Milk and Dairy
3
Products: Towards Spin-Chromatography?
4
5
Christina Papaemmanouil, Constantinos G. Tsiafoulis*§‡, Dimitrios Alivertis#,
6
Ouranios Tzamaloukas┴, Despoina Miltiadou┴, Andreas G. Tzakos, and Ioannis P.
7
Gerothanassis*
8
9
Section of Organic Chemistry and Biochemistry, Department of Chemistry, §NMR
10
Center, ‡ Laboratory of Analytical Chemistry, Department of Chemistry, #Department
11
of Biological Applications and Technology; University of Ioannina, Ioannina GR-451
12
10, Greece
13
┴
14
University of Technology, P. O. Box 50329, Limassol 3603, Cyprus
Department of Agricultural Sciences, Biotechnology and Food Sciences, Cyprus
15
16
1 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 2 of 26
17
ABSTRACT: We report a rapid, direct and unequivocal spin-chromatographic
18
separation and identification of minor components in the lipid fraction of milk and
19
common dairy products with the use of selective 1D TOCSY NMR experiments. The
20
method allows the complete backbone spin-coupling network to be elucidated even in
21
strongly overlapped regions and in the presence of major components with 4x102 to
22
3x103 stronger NMR signal intensities. The proposed spin chromatography method
23
does not require any derivatization steps for the lipid fraction, is selective with
24
excellent resolution, is sensitive with quantitation capability and compares favorably
25
with 2D TOCSY and GC-MS methods of analysis. The results of the present study
26
demonstrated that the 1D TOCSY NMR spin-chromatography method can become a
27
procedure of primary interest in food analysis and generally in complex mixture
28
analysis.
29
30
KEYWORDS: lipid fraction, totally correlated NMR spectroscopy (TOCSY), spin-
31
chromatography, dairy products
32
33
2 ACS Paragon Plus Environment
Page 3 of 26
Journal of Agricultural and Food Chemistry
34
INTRODUCTION
35
Separation and identification of components from complex mixtures has occupied a
36
central role in the field of natural products and food chemistry research. The classical
37
protocol for the investigation of complex mixtures has been to apply various
38
chromatographic techniques, to isolate a certain amount of a pure product and to
39
identify its structure using various spectroscopic techniques1 and/or the use of
40
specialized hyphenated spectroscopic techniques.2,3 On the other hand, NMR
41
spectroscopy is increasingly used as an analytical tool for identification and
42
quantification of low molecular weight metabolites on unfractionized biological
43
fluids, natural product extracts and food samples.1,4-12
44
Milk lipids are very important as they confer distinctive textural, nutritional and
45
organoleptic properties on dairy products. Recent studies have reported that the
46
consumption of saturated fatty acids has been linked to increased risk of
47
cardiovascular disease, whereas the consumption of milk conjugated linoleic acids
48
(CLA) has beneficial effects and this issue is still subject to numerous studies.13
49
Accurate analysis, therefore, of minor lipids is important for determining the nutritive
50
value and preparing nutritional labeling materials for particular function or
51
application. The analysis of minor lipids, however, is extremely challenging and
52
complex since it can be very slow and laborious and may require various preparation
53
and analysis steps.14
54
The selective 1D TOCSY experiment has become an important NMR technique for
55
establishing 1H-1H connectivity via scalar coupling in small and medium-size
56
molecules15,16 and in the analysis of mixtures of related compounds.17 However, the
57
method has found limited application in food matrices; to the best of our knowledge
3 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 4 of 26
58
few studies have, so far, been published, among them in the metabolomics analysis of
59
amino acids in honey18 and in mango juice.19 In the present study we report, for the
60
first time, the direct identification of six minor species: (9-cis, 11-trans) 18:2 and (9-
61
trans, 11-trans) 18:2 conjugated linoleic acid (CLA) isomers, caproleic acid, glycerol
62
in 1,2 diglyceride (1,2 DAG), in 1 monoglyceride (1-MAG), and in 2 monoglyceride
63
(2-MAG) in the lipid fraction of milk and halloumi cheese, without any derivatization
64
steps, with the use of a spin- chromatography procedure based on the spin diffusion
65
process of selective 1D TOCSY experiment.
66
67
MATERIALS AND METHODS
68
Materials. Conjugated (9-cis, 11-trans) 18:2 linoleic acid, purity ≥ 96% (HPLC),
69
conjugated (9-trans, 11-trans) 18:2 linoleic acid, purity ≥ 98% (HPLC), were
70
purchased from Fluka. Caproleic acid, purity ≥ 96%, was purchased from Sigma -
71
Aldrich, chloroform and methanol (analytical grade) were obtained from Fisher
72
Scientific (U.K.), CDCl3 (99.8%) from Deutero (Kastellaun, Germany) and the 37
73
FAME standard mix from Sigma-Aldrich.
74
Sample Preparations. The lipid fractions of milk samples were prepared as
75
previously described.20 The lipid fractions of halloumi cheese were prepared as
76
follows: cheese samples were frozen in liquid N2 and pulverized in a ceramic mortar.
77
After lyophilization for two days, 300 mg of cheese was used for the extraction of the
78
lipid phase, using the Bligh and Dyer method, as previously described.20
79
NMR Instrumentation. NMR experiments were performed on a Bruker AV500
80
spectrometer (Bruker Biospin, Rheinstetten, Germany) using the Topsin 2.1 suite. The
4 ACS Paragon Plus Environment
Page 5 of 26
Journal of Agricultural and Food Chemistry
81
1D TOCSY experiments were carried out using standard Bruker pulse program
82
(selmlgp). A shaped pulse length of 20 ms for selective excitation was used followed
83
either by a MLEV-17 TOCSY spin lock18 or by applying the DIPSI-2 pulse train and
84
by incorporating a z-filter before acquisition21 for the suppression of artifacts. The
85
spin-lock was adjusted to 7.1 KHz, corresponding to a low power 90o pulse of 35 µs;
86
this allows safe operation without problems of significant heating of the samples with
87
spin-lock times up to 400 ms.
88
GC-MS Analysis. Fatty acid methyl esters (FAME’s) were prepared by trans-
89
esterification with methanolic potassium hydroxide according to the ISO 15884:2002
90
method22 as previously described.23 Fatty acid profiles were generated by analyzing
91
the FAME samples on a GC-MS-QP 2010 Plus Gas Chromatography Mass
92
Spectrometer (Shimadzu, Duisburg, Germany) equipped with a HT 280 T auto
93
sampler (HTA, Brescia, Italy). Details of the GC-MS analysis are given
94
elsewhere.20,23
95
96
RESULTS AND DISCUSSION
97
1D TOCSY of Model Compounds – Effects of Mixing Time
98
Figure 1 illustrates a series of selective 1D TOCSY spectra of the model (9-cis, 11-
99
trans) 18:2 CLA isomer where the H11 olefinic proton (δ= 6.27 ppm)20,24,25 has been
100
selected for excitation using a range of mixing times and, thus, affecting the number
101
of transfers within the spin system.26 For a mixing time τm = 33 ms, the selective 1D
102
TOCSY transfers magnetization to its J-coupled partners within the conjugated
103
olefinic protons and the C(13)H2 protons (Figure 1(b)). Using longer mixing times,
5 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 6 of 26
104
the magnetization is transferred throughout the full spin system and, for τm values of
105
200 and 400 ms, resulted in the complete analysis and structure elucidation of the
106
compound (Figure 1(d), (e)). Figure S1 illustrates a similar series of selective 1D
107
TOCSY spectra of the (9-trans, 11-trans) 18:2 CLA isomer where the H10, H11 (δ=
108
5.97 ppm) protons have been selected. Again, the 1D TOCSY experiment allows
109
structural information to be extracted in a time-efficient manner and with high
110
spectral resolution.
111
112
Method Application in the Lipid Fraction of Milk and Dairy Products
113
An important application of the 1D experiment is the selective excitation of a
114
proton sub spectrum belonging to a single chemical component contained in a
115
complex mixture, thus providing a form of spin-chromatography. The selective
116
excitation of a suitable “target” resonance of the compound of interest can then reveal
117
the whole spin system, even if the 1D 1H NMR spectrum is heavily overlapped in the
118
region of the other peaks in the selected spin coupling network. Figure 2(a) illustrates
119
a typical 500 MHz 1H NMR experiment of the lipid fraction of a lyophilized halloumi
120
cheese sample in CDCl3. Selective TOCSY excitation, with 400 ms mixing time, of
121
the H11 proton of the (9-cis, 11-trans) 18:2 CLA isomer (Figure 2(b)), illustrates
122
effective magnetization transfer from H11 to H2 and H11 to H18. Therefore, its
123
structure becomes amenable to detailed analysis although signals from H2 to H8 and
124
H12 to H18 protons are completely hidden in a conventional 1D 1H NMR spectrum
125
under the resonances of the abundant components with 4x102 to 3x103 stronger
126
intensities.
6 ACS Paragon Plus Environment
Page 7 of 26
Journal of Agricultural and Food Chemistry
127
Similar experiments were performed with caproleic acid (Figure 2(c)). Selective
128
TOCSY excitation of the H10a protons at 4.97 ppm with τm = 400 ms resulted in the
129
effective magnetization transfer throughout the complete proton spin system, although
130
the signals from H8 to H2 are completely hidden in the conventional 1D 1H NMR
131
spectrum. Selective TOCSY excitation of the doublet at 3.72 ppm reveals the
132
complete spin system of the glycerol moiety in 1,2 DAG at 5.20 ppm (2’-CHOCO),
133
4.28 ppm and 4.13 ppm (1’b, 1’a-CH2OCO, respectively) (Figure 2(d)).
134
Figure S2 illustrates a similar series of selective 1D TOCSY spectra of the lipid
135
fraction of lyophilized milk sample. Again the 1D TOCSY allows the analysis and
136
structure elucidation of minor compounds in a time-efficient manner and with high
137
spectral resolution.
138
Comparison of the 1D TOCSY spectrum of the model (9-cis, 11-trans) 18:2 CLA
139
isomer with the one obtained from the lipid fraction of the lyophilized halloumi
140
cheese sample illustrates a significant difference of the C(2)H2 spin system (Figure 3).
141
In the model compound it appears as a triplet (δ= 2.33 ppm, 3J= 7.2 Hz) due to
142
coupling with the C(3)H2 protons while in the spectrum of the extract it appears at δ=
143
2.30 ppm with a complex multiplet pattern. This clearly demonstrates that the (9-cis,
144
11-trans) 18:2 CLA isomer in the extract exists predominantly as an ester and not as a
145
free acid. Due to the asymmetry of the sn2 carbon of the glycerol moiety the C(3)H2
146
protons become magnetically non equivalent, thus, resulting in a multiplet spin
147
pattern. This result is very important in investigating the hydrolysis products of the
148
ester linkage between an acyl group and the glycerol backbone. The resulting free
149
fatty acids and their catabolic products have been found to be among the primary
150
factors for the aroma of hard cheese where lipolysis reaches high levels.24
7 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 8 of 26
151
Figure 4 illustrates the great potential of the 1D TOCSY experiments in the case of
152
minor species with resonances in overcrowded spectral regions of the 1D 1H NMR
153
spectrum. The spin systems of the glycerol moieties in both 2-MAG (Figure 4(b1),
154
(b2)) and 1-MAG (Figure 4(c1), (c2)) were clearly resolved and provided an
155
unequivocal assignment of both species although the 1-MAG resonances are strongly
156
overlapped in the region of 3.75 to 3.55 ppm (Figure 4(a)).
157
The excellent selectivity of the 1D TOCSY is demonstrated also in the case of
158
strongly overlapped resonances of the 18:2 CLA geometric isomers. Figure S3
159
illustrates a selected 1H NMR region of the (9, 11) 18:2 CLA resonances of the lipid
160
fraction of a lyophilized halloumi cheese sample. The apparent triplet at 5.92 ppm
161
(3J= 10.9 Hz) has been assigned to the H10 olefinic proton of the (9-cis, 11-trans)
162
18:2 CLA isomer and the strongly overlapped minor peak at 5.97 ppm to the
163
composite signal of the H10 and H11 protons of the (9-trans, 11-trans) 18:2 CLA
164
isomer. Selective 1D TOCSY of the resonance at 5.97 ppm clearly demonstrates
165
magnetization transfer to H9 and H12 resonances at 5.54 ppm which are not
166
overlapped with other resonances of the lipid fraction (Figure S3(c)) and, thus, can be
167
used for the assignment of the (9-trans, 11-trans) 18:2 CLA isomer. A limitation of
168
the method, however, is that since the identification is based on chemical shifts, the
169
length of the fatty acid and the position of the unsaturation remains ambiguous. For
170
instance the 1H NMR spectrum of (9-cis, 11-trans) 18:2 has almost identical chemical
171
shifts and J-couplings as that from (9-cis, 11-trans) 20:2 and (11-cis, 13-trans) 20:2.
172
Figure S4 illustrates a comparison of the 1D TOCSY spectrum of Figure 2(b) with
173
the respective extracted column and row in the 2D TOCSY experiment. It is evident
174
that the 1D TOCSY: (i) increases significantly the digital resolution since in the 2D
175
TOCSY it is limited by the number of points taken in the indirect dimension, (ii) 8 ACS Paragon Plus Environment
Page 9 of 26
Journal of Agricultural and Food Chemistry
176
removes dynamic range issues from the spectrum; (iii) the whole spin system can be
177
excited even in the region of strong signal overlapping with major components with
178
4x102 to 3x103 stronger NMR signal intensities and (iv) decreases the experimental
179
time.
180
181
Quantification using 1D TOCSY – Comparison with 1D 1H NMR and GC-MS
182
Method of Analysis
183
The excellent sensitivity and digital resolution of the 1D TOCSY experiment
184
allows the quantification of minor components. The quantitative analysis was based
185
on the standard addition method. Known amounts of caproleic acid were added to the
186
sample and the respective spin chromatograms were recorded with the selective
187
excitation of the H9 proton of the caproleic acid. The pulse repetition time
188
(acquisition time + relaxation delay) was set at 5xT1 (~16 s) of the long relaxation
189
time of the H9 and H10 protons (~3.2 s) of the caproleic acid. The results were
190
compared with those obtained by the use of 1D 1H NMR quantitation.
191
The effect of the mixing time on the quantitation results was studied in detail.
192
Several mixing times were used, more specific 33, 100 and 200 ms (τm= 33 ms
193
corresponds to the value of ½ 3J, where 3J is the coupling of the H9 and H10 protons).
194
The sensitivity of the 1D TOCSY method was higher using smaller mixing time
195
values but at the expense of the quantitation accuracy. Longer mixing times resulted
196
in a moderate decrease of sensitivity, approx. by a factor of two, however, a
197
significantly improved accuracy of the method was achieved (the relative error was
198
calculated 31 % for τm= 33 ms, due to twisted lineshapes,27 whereas only 2 % for τm=
199
200 ms). Moreover for τm= 200 ms the sensitivity and accuracy of the method was 9 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 10 of 26
200
almost equal using either the H9 or the H10 signals (data not shown). It should be
201
pointed out that one of the advantages of the spin chromatographic procedure is the
202
ability to quantify the analyte of interest either using the excited proton or by using a
203
nearby proton where the magnetization was transferred.
204
1D TOCSY quantitation was achieved following the procedure described by
205
Sandusky et al.28 Table 1 provides the quantification data obtained for the milk
206
samples using the 1D TOCSY procedure. A concentration value (in tube) of
207
1.02±0.03 mmol L-1 was determined using the 1D TOCSY and the H9 proton
208
integration (Figure 5 and Figure S5) compared to 0.97±0.02 mmol L-1 as determined
209
by using the conventional 1D 1H NMR with internal standard. For the second milk
210
sample, the respective results were 1.92±0.06 and 1.84±0.06 mmol L-1. The results
211
demonstrate the excellent agreement between the 1D TOCSY method with the
212
conventional 1D 1H NMR method.
213
The limit of detection (LD) for the spin chromatographic quantitation of the
214
caproleic acid was calculated by the use of two different methods, based on the
215
calibration function29 or the SNR method.30,31 By means of the calibration function,29
216
a calibration curve of the standard addition method was used; the LD was found 0.07
217
± 0.01 mmol L-1 (in tube) or 0.01 mg g-1 for the milk sample. Using the SNR method
218
the LD was calculated by applying the 3xC/(S/N) equation in a standard sample of
219
caproleic acid, where C represents the concentration of the analyte as determined by
220
1
221
the signal-to-noise value of the respective 1D TOCSY spectra was calculated. For the
222
concentration levels of 0.97, 2.04 and 2.98 mmol L-1 of caproleic acid (in tube) the
223
3xC/(S/N) value was calculated 0.10, 0.11 and 0.08 mmol L-1 using the 1D TOCSY
224
spectra, and 0.004, 0.007 and 0.010 mmol L-1 using the 1D 1H NMR spectra,
H NMR (data were treated without using a line broadening exponential function) and
10 ACS Paragon Plus Environment
Page 11 of 26
Journal of Agricultural and Food Chemistry
225
respectively; thus, the LD value of caproleic acid is 0.10 mmol L-1 (in tube) or 0.01 ±
226
0.001 mg g-1 for the milk sample and 0.010 mmol L-1 (in tube) or 0.001 ± 0.0001 mg
227
g-1 for 1D TOCSY and 1D 1H NMR methods, respectively.
228
The quantitative results of the spin chromatography and 1H NMR were compared
229
with those obtained with the use of the GC-MS method of analysis (ISO
230
15884:2002).22 The results are in accordance with the GC-MS method of analysis;
231
caproleic acid was found to be 0.28 % and 0.68% of the total lipids when calculated
232
by GC-MS and was measured 0.26 % and 0.74 % of the total lipids when calculated
233
by 1D 1H NMR for the first and second milk sample, respectively.
234
The proposed selective 1D TOCSY spin-chromatographic separation procedure,
235
therefore, is an excellent technique in mixture analysis of minor components, through
236
a careful selection of the spin system to be excited. The method might become of
237
primary interest in food research including targeting metabolomics32 since: (i) it is
238
rapid, selective and nondestructive, (ii) it allows the chemical identification of minor
239
components even in strongly overlapped spectral regions, (iii) does not require
240
derivatization steps and (iv) allows the quantification of analytes of interest.
241
242
AUTHOR INFORMATION
243
Corresponding Authors
244
*(I.P.G.) E-mail:
[email protected]; Phone: +30 26510 08389
245
*(C.G.T) E-mail:
[email protected]; Phone: +30 26510 08315
246
Notes
11 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
247
Page 12 of 26
The authors declare no competing financial interest.
248
249
ACKNOWLEDGMENTS
250
This work was supported by the Cyprus Research Promotion Foundation, European
251
Regional Development Fund and Charalambides- Christies Ltd dairy industry. Thanks
252
are given to the anonymous reviewers for critical and constructive criticisms.
253
254
Supporting Information Available
255
Figure S1: 500 MHz 1H NMR and 1D TOCSY spectra for several mixing times of (9-
256
trans, 11-trans) 18:2 conjugated linoleic acid; Figure S2: 500 MHz 1H NMR and 1D-
257
TOCSY spectra of the lipid fraction of a lyophilized milk sample; Figure S3: Selected
258
regions of 500 MHz 1H NMR and 1D TOCSY spectra for (9-cis, 11-trans) and (9-
259
trans, 11-trans) 18:2 CLAs of the lipid fraction of a lyophilized cheese sample; Figure
260
S4: Selected regions of 500 MHz 2D TOCSY and 1D TOCSY spectra of the lipid
261
fraction of a lyophilized cheese sample; Figure S5: 1D TOCSY quantitation
262
procedure displaying the 1D TOCSY spectra for successive addition of caproleic acid
263
in a milk sample.
264
This information is available free of charge via the Internet at http: //pubs.acs.org
12 ACS Paragon Plus Environment
Page 13 of 26
Journal of Agricultural and Food Chemistry
265
REFERENCES AND NOTES
266
(1) Novoa-Carballal, R.; Fernandez-Megia, E.; Jimenez, C.; Riguera, R. NMR
267
methods for unravelling the spectra of complex mixtures. Nat. Prod. Rep. 2011,
268
28, 78–98.
269 270
(2) Albert, K. On-line LC-NMR and related techniques, John Wiley & Sons, Hoboken, NJ, USA, 2002.
271
(3) Wolfender, J.L.; Marti, G.; Thomas, A.; Bertrand, S. Current approaches and
272
challenges for the metabolite profiling of complex natural extracts. J.
273
Chromatogr. A 2014, 1382, 136-164.
274
(4) Lewis, I.A.; Schommer, S.C.; Hodis, B.; Robb, K.A.; Tonelli, M.; Westler, W.M.;
275
Sussman, M.R.; Markley, J.L. Method for determining molar concentrations of
276
metabolites in complex solutions from two-dimensional 1H-13C-NMR spectra.
277
Anal. Chem. 2007, 79, 9385-9390.
278 279
(5) Spyros, A.; Dais, P.
31
P NMR spectroscopy in food analysis. Progr. NMR
Spectrosc. 2009, 54, 195-207.
280
(6) Charisiadis, P.; Exarchou, V.; Troganis, A.N.; Gerothanassis, I.P. Exploring the
281
“forgotten” –OH NMR spectral region in natural products. Chem. Commun.
282
2010, 46, 3589-3591.
283
(7) Manjunatha Reddy, G.N.; Caldarelli, S. Maximum-quantum (maxQ) NMR for the
284
speciation of mixtures of phenolic molecules. Chem. Commun. 2011, 47, 4297-
285
4299.
13 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
286
Page 14 of 26
(8) Charisiadis, P.; Primikyri, A.; Exarchou, V.; Tzakos, A.; Gerothanassis, I. P. 1
287
Unprecedented ultra-high-resolution hydroxy group
H NMR spectroscopic
288
analysis of plant extracts. J. Nat. Prod. 2011, 74, 2462–2466.
289
(9) Charisiadis, P.; Tsiafoulis, C.G.; Exarchou V.; Tzakos, A.; Gerothanassis, I. P.
290
Rapid and direct low micromolar NMR method for the simultaneous detection of
291
hydrogen peroxide and phenolics in plant extracts. J. Agric. Food Chem. 2012,
292
60, 4508–4513.
293
(10) Robinette, S.L.; Brüschweiler, R.; Schroeder, F.C.; Edison, A.S. NMR in
294
metabolomics and natural products research: Two sides of the same coin. Acc.
295
Chem. Res. 2012, 45, 288-297.
296 297
(11) Mannina, L.; Sobolev, A.P.; Viel, S. Liquid state 1H high field NMR in food analysis. Progr. NMR Spectrosc. 2012, 66, 1-39.
298
(12) Martin-Pastor, M. Experiments for the editing of singlet peaks and simplification
299
of 1H NMR spectra of complex mixtures. J. Agric. Food Chem. 2014, 62, 1190–
300
1197.
301 302
(13) Dilzer, A.; Park, Y. Implication of conjugated linoleic acid (CLA) in human health. Crit. Rev. Food Sci. 2012, 52, 488-513.
303
(14) Shahidi, F.; Wanasundara, P. K. J. P. D. in: Akoh, C. C.; Min, D. B. (Eds). Food
304
Lipids, Chemistry, Nutrition and Biotechnology, Marcel Decker Inc., New York;
305
2012.
306 307
(15) Dalvit, C.; Bovermann, G. Pulsed field gradient one-dimensional NMR selective ROE and TOCSY experiments. Magn. Reson. Chem. 1995, 33, 156-159. 14 ACS Paragon Plus Environment
Page 15 of 26
Journal of Agricultural and Food Chemistry
308
(16) Parella, T. High - quality 1D spectra by implementing pulsed-field gradients as
309
the coherence pathway selection procedure. Magn. Reson. Chem. 1996, 34, 329-
310
347.
311
(17) Sharman, G.J. Development of a selective TOCSY experiment and its use in
312
analysis of a mixture of related compounds. Chem. Commun. 1999, 14, 1319-
313
1320.
314
(18) Sandusky, P.; Raftery, D. Use of semiselective TOCSY NMR experiments for
315
quantifying minor components in complex mixtures: application to the
316
metabonomics of amino acids in honey. Anal. Chem. 2005, 77, 2455-2463.
317
(19) Koda, M; Furihara, K.; Wei, F.; Moyakawa, T. Metabolic discrimination of
318
mango juice from various cultivars by band- selective NMR spectroscopy. J.
319
Agric. Food Chem. 2012, 60, 1158–1166.
320
(20) Tsiafoulis, C. G.; Skarlas, T.; Tzamaloukas, O.; Miltiadou, D.; Gerothanassis, I.
321
P. Direct nuclear magnetic resonance identification and quantification of
322
geometric isomers of conjugated linoleic acid in milk lipid fraction without
323
derivatization steps: overcoming sensitivity and resolution barriers. Anal. Chim.
324
Acta 2014, 821, 62–71.
325
(21) Cavanagh, J.; Rance, M. Supression of cross-relaxation effects in TOCSY spectra
326
via a modified DIPSI-2 mixing sequence. J. Magn. Reson. 1992, 96, 670–678.
327
(22) ISO, International standard. Milk fat – Preparation of fatty acid methyl esters.
328
ISO 15884:2002 (IDF 182:2002). International organization of standardisation,
329
Geneva, Switzerland, 2002, 6.
15 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 16 of 26
330
(23) Tzamaloukas, O.; Ordford, M.; Miltiadou, D.; Papachristoforou, C. Partial
331
suckling of lambs reduced the linoleic and conjugated linoleic acid contents of
332
marketable milk in Chios ewes. J. Dairy Sci. 2015, 98, 1739-1749.
333
(24) Scano, P.; Anedda, R.; Melis, M.P.; Dessi, M.A.; Lai, A.; Roggio, T. 1H- and
334
13
335
Pecorino Sardo cheese. J. Am. Oil Chem. Soc. 2011, 88, 1305-1316.
C-NMR characterization of the molecular components of the lipid fraction of
336
(25) Prema, D.; Pilfold, J. L.; Krauchi, J.; Church, J. S.; Donkor, K.K.; Cinel, B.
337
Rapid determination of total conjugated linoleic acid content in select Canadian
338
cheeses by 1H NMR spectroscopy. J. Agric. Food Chem. 2013, 61, 9915–9921.
339
(26) Sachleben, J. R.; Yi, R.; Volden, P. A.; Conzen, S.D. Aliphatic chain length by
340
isotropic mixing (ALCHIM): determining composition of complex lipid samples
341
by 1H NMR spectroscopy. J. Biomol. NMR 2014, 59, 161-173.
342
(27) An effective elimination of the twisted lineshapes (anti-phase components, ZQ
343
artefacts) for short τm values can be achieved with the use of the ZQ-filter
344
experiment (Thrippleton, J.M.; Keeler, J. Elimination of zero-quantum
345
interference in two-dimensional NMR spectra, Angew. Chem., Int. Ed. 2003, 42,
346
3938 –3941).
347
(28) Sandusky, P.; Amponsah, E.A.; Raftery, D. Use of optimized 1D TOCSY NMR
348
for improved quantitation and metabolomic analysis of biofluids, J. Biomol. NMR
349
2011, 49, 281-290.
350 351
(29) Danzer, K. Analytical Chemistry, Theoretical and Metrological Fundamentals; Springer- Verlag: Berlin, 2007; pp.74, 147.
16 ACS Paragon Plus Environment
Page 17 of 26
Journal of Agricultural and Food Chemistry
352
(30) Tsiafoulis. C.G.; Exarchou, V.; Tziova, P.P.; Bairaktari, E.; Gerothanassis, I.P.;
353
Troganis, A.N. A new method for the determination of free L-carnitine in serum
354
samples based on high field single quantum coherence filtering 1H-NMR
355
spectroscopy. Anal. Bioanal. Chem. 2011, 399, 2285-2294.
356
(31) Maniara, G.; Rajamoorthi, K.; Rajan, S.; Stockton, G. Method performance and 1
validation for quantitative analysis by
358
Applications to analytical standards and agricultural chemicals. Anal. Chem.
359
1998, 70, 4921-4928.
360 361
H and
31
357
P NMR spectroscopy.
(32) Sundekilde, U. K.; Larsen, L. B.; Bertram, H. C. NMR-based milk metabolomics. Metabolites 2013, 3, 204–222.
362
363
17 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 18 of 26
364
FIGURE CAPTIONS
365
Figure 1. (a) 500 MHz 1D NMR spectrum of 20 mM solution of the (9-cis, 11-trans)
366
18:2 conjugated linoleic acid in CDCl3 (T= 298 K, acquisition time= 4.3 s, relaxation
367
delay= 5 s, number of scans= 256, experimental time ~25 min); (b)-(e) selective 1D
368
TOCSY spectra of the above solution using a mixing time of τm= 33 ms (b), 70 ms
369
(c), 200 ms (d), and 400 ms (e). The asterisk denotes the selected H11 resonance
370
which was excited. For (b)-(e) the magnetization transfer network is illustrated.
371
Figure 2. Spin chromatogram of the lipid fraction of a lyophilized cheese sample in
372
CDCl3: (a) 500 MHz 1H NMR spectrum of the lipid fraction of a lyophilized cheese
373
sample in CDCl3 (298 K, number of scans= 256, acquisition time= 4.3 s, relaxation
374
delay= 5 s, total experiment time ~25 min). The major lipid resonances are denoted
375
(sn1, sn2 and sn3 indicate the stereospecific numbering of esterified glycerol). The
376
insert shows x512 magnification of the spectrum in order to display resonances from
377
the 18:2 CLA and other minor species. (b)-(d) 1D TOCSY spectra of 2(a) with τm=
378
400 ms (ns= 256, total experiment time ~25 min). The asterisks denote the resonances
379
which were excited by the use of a selective pulse.
380
Figure 3. Selected regions of: (a) Figure 2(a); (b) 1D TOCSY spectrum of Figure
381
2(b); (c) 1D TOCSY of the model (9-cis, 11-trans) 18:2 CLA isomer. In (b) and (c)
382
the selective excitation pulse was set on the H11 proton (δ= 4.92 ppm). The insert in
383
(b) shows x16 magnification of the spectrum in order to display the C(2)H2COOR
384
resonances.
385
Figure 4. Selected regions of: (a) Figure 2(a); (b) 1D TOCSY spectrum which
386
demonstrates the spin system of the glycerol moiety in 2-MAG (in (b1) and (b2) the
387
selective excitation pulse was set on the 1’,3’ CH2OH (δ= 3.82 ppm) and 18 ACS Paragon Plus Environment
Page 19 of 26
Journal of Agricultural and Food Chemistry
388
2’ CHOCOR (δ= 4.92 ppm) peaks in 2-MAG, respectively); (c) 1D TOCSY spectrum
389
of the spin system of the glycerol moiety in 1-MAG (in (c1) and (c2) the selective
390
excitation pulse was set on the 3’ CH2OH (δ= 3.67 ppm) and 2’ CH2OH (δ= 3.92
391
ppm) peaks in 1-MAG, respectively).
392
Figure 5. Quantification of caproleic acid (CA) in the lipid fraction of a lyophilized
393
milk using the 1D TOCSY method (see also Figure S5).
394
19 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 20 of 26
Table 1: Results obtained using 1D TOCSY NMR, conventional 1D 1H NMR and GC-MS methods for the determination of the caproleic acid in two milk samples. Sample
Analyte / Units
1D
1
H NMRa
TOCSYa 1
Caproleic acid/mM
1.02±0.03
Caproleic acid / %
Relativeb
GC-MS
deviation (%) 0.97±0.02a
Relativec deviation (%)
5.15
0.26±0.01
0.28
-7.14
0.68
8.82
of the lipid fraction 2
Caproleic acid/mM % of the lipid
1.92±0.06
1.83±0.06a
1.75
0.74±0.01
fraction a
Results are expressed as mM of caproleic acid (in tube; standard deviation (n=3)). bResults are
expressed as 100 x [ (1D TOCSYvalue)- (1D 1H NMRvalue)] /(1D 1H NMRvalue). cResults are expressed as 100 x [(1D 1H NMRvalue) -(GC-MSvalue)] /(GC-MSvalue).
20 ACS Paragon Plus Environment
Page 21 of 26
Journal of Agricultural and Food Chemistry
Figure 1
21 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 22 of 26
Figure 2
22 ACS Paragon Plus Environment
Page 23 of 26
Journal of Agricultural and Food Chemistry
Figure 3
23 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 24 of 26
Figure 4
24 ACS Paragon Plus Environment
Page 25 of 26
Journal of Agricultural and Food Chemistry
Figure 5
25 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
TOC Graphic
ACS Paragon Plus Environment
Page 26 of 26